Cellular analysis and research often requires the manipulation of small particles, including cells, cell aggregates, cell organelles, stem cells, nucleic acids, bacteria, protozoans, viruses, and/or other micro- and/or nano-particles. Typically, the small particles to be manipulated have a dimension (e.g., diameter) ranging from approximately 0.1 micrometer to approximately several hundred micrometers, for example from approximately 1 micrometer to approximately 100 micrometers, or, for example, from approximately 5 micrometers to approximately 10 micrometers. By way of example only, mammalian cells have a diameter ranging from about 5 micrometers to about 100 micrometers and a lymphocyte may be about 10 micrometers in diameter. In some cases, groups of particles (e.g., cells, stem cells, etc.) may be separated from other particles. The dimension of a group of particles may be as large as about 100 micrometers.
Various devices and methods have been used to manipulate small particles so as to identify, discriminate, sort, characterize, quantitate, observe, move, collect, and/or otherwise manipulate the small particles, such as, for example, live stem cells. For example, microfluidic devices that rely on pressure-driven flow to separate cells, for example sperm cells from epithelial cells, have been utilized. This technique is a passive technique and relatively cost effective for cell sorting, however, the operation protocol is craft sensitive and must be determined on an application by application basis In other words, because microfluidic devices rely on pressure-induced flow to separate cells by virtue of their size and flow rate, appropriate operational conditions must be determined on a trial-and-error basis, since nontarget and target cells may be of substantially the same size. Moreover, due to the relatively narrow microfluidic channels and cross-junctions present in such microfluidic devices, care must be taken to avoid rupturing cells while forcing them through the small passageways.
Flow cytometers, including fluorescence activated sorters, for example, are relatively complex optics-based instruments that serially analyze and isolate fluorescently-labeled cells from a flowing stream of fluid. One such device of this class has been used to manipulate Escherichia coli cells. This sorting device comprises a narrow capillary T-shaped junction connected to three reservoirs at each end for receiving aqueous sample, collection, and waste, respectively. The device relies on electro-osmotic flow (EOF) for cell transport and a preset fluorescence threshold to trigger the switching of EOF direction at the T-shaped junction, thereby resulting in cell sorting. A modified version of the device, a microfluidic cell sorting device, has been utilized to sort stably transfected HeLa cells. This modified version relies on pressure-driven flow and a focused laser spot at the junction to deflect and reroute cells by optical force gradient (optophoresis) to a collection reservoir.
The reliance on lasers and other optics contributes to relatively high fabrication costs of some flow cytometers. Further, when using such devices, it may be necessary to simultaneously optimize the optical, fluidic, electronic, and computer systems, and efficiency may be reduced.
Aside from being relatively complex systems and having relatively high fabrication costs, disadvantages of fluorescence activated sorting techniques may include, among others, limited run time due to ion depletion of the sample solution as a result of electro-osmosis and clogging of small orifices and other passageways. Regarding the latter, the size of the orifices at the T-shaped junction are typically relatively small, for example about 3 to about 10 microns. Thus, depending on the types of particles (e.g., cells) being manipulated, some particles may be too large to pass through the junction. Moreover, passive adsorption of proteins and/or other material may occur on the surfaces at the junction, causing a build up of such materials on the surface and potentially result in clogging of the device. Further, if polydimethylsiloxane (PMDS) is used to fabricate a flow cytometer, performing a surface modification on PMDS in order to reduce nonspecific adsorption of biomolecules poses challenges.
Another potential drawback may include the use of dyes to label for recognition various cells of interest. In the case of stem cells, for example, using dyes and other labeling techniques potentially could harm and/or otherwise stress the cells. Similarly, in cases where relatively high intensity lasers are used, such lasers could harm and/or cause stress to the cells. Proliferating potentially stressed cells and possibly implanting the proliferated cells back into a patient could possibly pose potential health and/or other risks. Moreover, the sorting throughput of flow cytometers may be limited in that such devices typically operate on a cell-by-cell manipulation basis. Although, the manipulation of each cell occurs relatively rapidly, due to the cell-by-cell manipulation scheme, the amount of time it may take to manipulate all of the cells in a sample may be relatively large.
Other techniques for manipulating small particles include the use of a dielectrophoretic force. Dielectrophoresis (DEP) refers to the motion imparted on uncharged objects as a result of polarization induced by a spatially nonuniform electric field. An analytical expression of the dielectrophoretic force, {right arrow over (F)}DEP, acting on a particle (T. B. Jones, Electromechanics of Particles, Cambridge University Press, 1995) is set forth below:
                    F        ->            DEP        =          2      ⁢                          ⁢      π      ⁢                          ⁢              r        3            ⁢              ɛ        m            ⁢              α        r            ⁢                        ∇          ->                ⁢                  (                                    E              ->                        RMS            2                    )                      ,          ⁢            α      r        ≡          Re      (                                    ɛ            p            *                    -                      ɛ            m            *                                                ɛ            p            *                    +                      2            ⁢                                                  ⁢                          ɛ              m              *                                          )      
In the above equation, r is the radius of the particle, the factor in parentheses in the first line of the equation is the RMS value of the electric field, and αr is the real part of the Clausius-Mosotti factor which relates the complex permittivity of the object ∈p and the complex permittivity of the medium ∈m. The star (*) denotes that the complex permittivity is a complex quantity. The Clausius-Mosotti factor may have any value between 1 and −½, depending on the applied AC frequency and the complex permittivity of the object and medium. If it is less than zero, the dielectric force is negative and the particle moves toward a lower electric field. If the Clausius-Mosotti factor is greater than zero, the dielectric force is positive and the particle moves toward a stronger electric field. In other words, if the object (e.g., particle, cell, etc.) is more polarizable than its surroundings, it may be pulled toward relatively strong field regions (“positive DEP”) and if it is less polarizable, it may be pulled toward relatively weak field regions (“negative DEP”).
If the particles are charged, then under DC current or low frequency AC current, electrophoresis (EP) occurs, instead of DEP. EP refers to the lateral motion imparted on charged objects in a nonuniform or uniform electric field.
DEP has been used to manipulate particles, such as cells, for example, via a traveling wave generated by a series of patterned electrodes lining up and charged with phase-shifted AC signals. The electrodes can be patterned in an independently controlled array to provide the traveling wave. For examples of such a technique, reference is made to Pethig et al., “Development of biofactory-on-a-chip technology using excimer laser micromachining,” Journal of Micromechanics and Microengineering, vol. 8, pp. 57-63, 1998, and Green et al., “Separation of submicrometer particles using a combination of dielectrophoretic and electrohydrodynamic forces,” Journal of Physics D: Applied Physics, vol. 31, L25-L30, 1998. In one technique, disclosed by Das et al., “Dielectrophoretic Segregation of Different Cell Types on Microscope Slides,” Anal. Chem. May 1, 2005, vol. 77, pp. 2708-2719, incorporated by reference herein, a glass slide is patterned with an electrode array in which the electric field frequency decreases in one direction along the length of the slide, which in turn results in a variation of generated DEP forces along the length of the slide. For other examples of the use of DEP particle manipulation via a traveling wave, reference is made to Hagendorn, et. al., “Traveling-wave dielectrophoresis of microparticles,” Electrophoresis, vol. 12, pp. 49-54, 1992 and Talary et al., “Electromanipulation and separation of cells using traveling electric fields,” J. Phys. D: Appl. Phys., vol. 29, pp. 2198-2203 (1996), the entire contents of both of which are incorporated by reference herein.
The use of DEP for separating differing cell types in a device wherein electrode arrays are used to create the nonuniform electric field also has been disclosed, for example, in U.S. Pat. No. 6,790,330 B2, which issued on Sep. 14, 2004, U.S. Pat. No. 6,641,708 B1, which issued on Nov. 4, 2003, and U.S. Pat. No. 6,287,832 B1, which issued on Sep. 11, 2001, the entire disclosure of each of which is incorporated by reference herein. These patents disclose various devices and methods relying on DEP induced by electrodes for cell separation.
Another technique for manipulating cells includes the use of optophoresis to manipulate cells in a surrounding medium, such as, for example, an aqueous suspension. Devices and methods utilizing optophoresis rely on a radiation pressure force generated by laser-induced optical gradient fields to capture and manipulate micrometer-scale particles in the aqueous suspension. Devices and methods relying on optophoresis and high intensity lasers to directly trap a single particle have been dubbed “optical tweezers.” For exemplary applications utilizing the principles of optical tweezers, reference is made to Ashkin et al., “Optical trapping and manipulation of single cells using infrared laser beams,” Nature, vol. 330, December 1987, pages 769-771; and Arai et al, “Tying a molecular knot with optical tweezers,” Nature, vol. 399, June 1999, pages 446-448, each of which is incorporated by reference herein.
Aside from the optical tweezers optophoretic technique, another technique employing optophroetic principles uses a fast-scan optophoresis device for recognizing, identifying, and quantifying one type of cells from among others. Such a technique is discussed, for example, in U.S. Application Publication No. 2002/0160470 A1, published Oct. 31, 2002; U.S. Application Publication No. 2005/0164372 A1, published Jul. 28, 2005; Hoo et al., “A Novel Method for Detection of Virus-Infected Cells Through Moving Optical Gradient Fields Using Adenovirus as a Model System,” Cytometry Part A, 58A, February 2004, pages 140-146; and Forster et al., “Use of moving optical gradient fields for analysis of apoptotic cellular responses in a chronic myeloid leukemia cell model,” Analytical Biochemistry, 327, 2004, pages 14-22, the entire disclosure of each of which is incorporated by reference herein.
In an embodiment, the fast-scan optophoresis device includes a CCD (charge couple device) camera and a coherent Nd-YAG 1064 nm laser beam, operating at 18.3 kW/cm2 at a focused point, scanning across the surface of a thin-layer cell in which a suspension of various types of particles (e.g., cells) are contained. Under a given set of conditions, all of the particles in the suspension are swept across the thin-layer cell by the laser beam until the laser beam's scanning speed reaches a threshold. Above that threshold, one type of particles escapes and is left behind the sweeping laser beam due to various forces, including drag forces, acting on that particle type. Software is used to measure the optophoretic distance that each particle travels and accumulated statistics may be used for identification and quantitation. This device has been used to analyze chronic myeloid leukemia cells and HeLa human ovarian carcinoma cells. Due to the use of high power lasers and focusing optics, manufacturing costs associated with such optophoretic scanning devices may be relatively high. Moreover, the use of high intensity lasers may potentially harm and/or otherwise stress the cells. In the case of stem cells, for example, that may be proliferated after being sorted and collected and then implanted into a patient, there may be a risk associated with such cell stressing.
Another more recently developed particle manipulation technique includes so-called “optoelectronic tweezers,” which have been used to attract or expel a plurality of small particles by application of an optically activated DEP force. In contrast to optical tweezers, optoelectronic tweezers can use a low power incoherent light source, for example, on the order of 1 μW/cm2, instead of the high intensity laser used by optical tweezers. By way of example, optoelectronic tweezers may utilize a light source that has a power approximately ten orders of magnitude less than that of the high intensity lasers typically employed in optical tweezers. In the optoelectronic tweezers technique, by projecting the low power incoherent light source onto a photoconductive surface, a liquid suspension containing various particles, e.g., cells, sandwiched between a patternless photoconductive surface and another patternless surface may be subject to a nonuniform electric field resulting from the illumination of the photoconductive layer. In turn, a dielectrophoretic force is created and acts on the particles. Particles may then be attracted by or repelled from the illuminated area depending upon, among other things, the particles' dielectric properties.
One device that employs the above-described principles includes a manipulation chamber comprising a top indium tin oxide transparent glass electrode, a bottom substrate coated with photoconductive material to complete the circuitry, and an aqueous layer containing particles of interest sandwiched between the surfaces. A focused incoherent light spot creates a nonuniform electric field by which the particles (e.g., live cells) in the aqueous sandwiched layer are manipulated based on their respective dielectric constants and sizes.
For further explanation of the operation principles of optoelectronic tweezers, including various devices and techniques employing those principles, reference is made to Pei Yu Chiou et al., “Massively parallel manipulation of single cells and microparticles using optical images,” Nature, vol. 436:21, July 2005, pages 370-372; PCT publication number WO 2005/100541, entitled “Optoelectronic Tweezers for Microparticle and Cell Manipulation,” which claims priority to U.S. Provisional Application No. 60/561,587, filed on Apr. 12, 2004; U.S. application Ser. No. 10/979,645, entitled “Surface Modification For Non-Specific Adsorption Of Biological Material,” filed Nov. 1, 2004, in the name of Aldrich Lau; and U.S. Provisional Application No. 60/692,528, entitled “Optoelectronic separation of biomolecules: Separation of dye-labeled DNA, RNA, proteins, lipids, terpenes, and polysaccharides,” filed Jun. 30, 2005, in the name of Aldrich Lau, the entire contents of each of which are incorporated by reference herein.
Conventional optoelectronic tweezers are typically employed by providing a manipulation chamber on a microscope stage and targeting predetermined cells of interest. Once the cells of interest are in view, the light source can be mapped onto the manipulation chamber and the predetermined cells can be captured. Thus, the existing devices use previsualization in order to capture known targets of interest.
Based on current techniques for manipulating small particles, including sorting, identifying, characterizing, quantifying, moving and/or otherwise manipulating small particles, it may be desirable to provide a technique for manipulating small particles that is relatively inexpensive to make and/or use and/or is disposable. It may be desirable to provide a manipulation device that is relatively easy to fabricate. For example, it may be desirable to provide a technique that may not require patterned electrodes, microchannels, capillary junctions, capillary orifices, relatively expensive lasers or optics, high power lasers, and/or other elements that are relatively expensive and/or intricate to fabricate. It also may be desirable to provide a device that relies on DEP to manipulate particles and achieves greater flexibility and control over modulation of the electric field than conventional device that utilize patterned electrodes. Moreover, it may be desirable to provide a particle manipulation technique that reduces potential clogging that can occur in device having relatively small junctions and/or orifices through which particles must pass.
Further, it may be desirable to provide a technique that achieves high sorting throughput, purity, and/or the recovery of undamaged (e.g., uncontaminated and/or unstressed) cells. It may further be desirable to provide a technique that achieves the recovery of live, unstressed mammalian cells. For example, it may be desirable to provide a technique that sorts stem cells from other cells, such as mouse feeder cells, and recovers the stem cells uncontaminated and/or unstressed. It also may be desirable to provide a particle manipulation technique that does not require the cells to be chemically labeled and/or exposed to high intensity laser radiation. Although it may be desirable to provide a manipulation technique that does not require chemical (e.g., including dyes and other fluorescence labeling), it also may be desirable to provide a manipulation technique that can work in conjunction with conventional detection methods, including the use of fluorescence signal detection, for example.
It may be desirable to provide a technique that permits visualization of the manipulation (e.g., sorting) of cells via a microscope, a camera, or other visualization tool.
It also may be desirable to provide a technique which selectively sorts cells based on various cell properties, such as, for example dielectric constant and size, and which may be automated. Moreover, it may be desirable to provide a technique that permits surface modification of the device, for example, to alter nonspecific and/or specific adsorption, and/or surface modification of the particles being manipulated. Regarding the former, surface modification of the device may be beneficial to reduce or enhance nonspecific adsorption of, for example, proteins, lipids, cells, and/or other biomolecules. Regarding the latter, it may be desirable to provide a technique that permits reversible surface modification of the particles so as to alter the particles' size, dielectric constant, polarity, and/or other properties.
It may also be desirable to improve upon existing devices that utilize optoelectronic tweezers principles in order to manipulate cells. For example, it may be desirable to provide a device that improves adhesion of the photoconductive and/or electrode layer to the glass substrate, improves robustness, and/or enables operation at a relatively low AC frequency or via direct current. It also may be desirable to reduce nonspecific adsorption of biomolecules. It also may be desirable to provide a device that enables surface modification of the substrates so as to, among other things, reduce nonspecific adsorption and permit the use of surface active agents (e.g., ligands, etc.) to differentiate particles.
Further, it may be desirable to utilize the principles associated with optoelectronic tweezers and/or other optoelectronic manipulation techniques and chambers in conjunction with existing manipulation techniques. In other words, it may be desirable to provide an optoelectronic manipulation chamber as an accessory to a microscope or portable medical device. It may also be desirable to provide a device that utilizes the principles of optoelectronic tweezers in combination with conventional manipulation techniques, such as for example, laser pressure catapulting, laser microdissection, laser microinjection, electroporation, microcapillaries, microdissector, microinjection, micromanipulators, piezoelectric microdissection, drug interaction/cell response, ion channel conductivity measurement (patch clamp), and/or other types of manipulation techniques. To achieve such a combination, it may be desirable to provide an optoelectronic manipulation chamber that permits insertion of an instrument or other external element into the liquid sample cavity containing the particles to be manipulated.
A further desirable aspect may include particle manipulation techniques that may be automated.